In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

In vitro proliferation and differentiation of hepatic oval cells and their potential capacity for intrahepatic transplantation

Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P=0.003, Studentt-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control=6.3±0.9; HOC=3.5±1.5; P=0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.

Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

The effect of hypoxia and reoxygenation in the response of mesangial cells to angiotensin II in vitro

INTRODUCTION: Mesangial cells (MC) may be involved in the glomerular alterations induced by ischemia/reperfusion injury. OBJECTIVE: To evaluate the response of immortalized MC (IMC) to 30 minutes of hypoxia followed by reoxygenation periods of 30 minutes (H/R30) or 24 hours (H/R24). METHODS: The intracellular calcium concentration ([Ca+2]i) was measured before (baseline) and after adding angiotensin II (AII, 10-5 M) in the presence and absence of glybenclamide (K ATP channel blocker). We estimated the level of intracellular ATP, nitric oxide (NO) and PGE2. RESULTS: ATP concentration decreased after hypoxia and increased after reoxygenation. Hypoxia and H/R induced increases in basal [Ca+2]i. AII induced increases in [Ca+2]i in normoxia (97 ± 9%), hypoxia (72 ± 10%) or HR30 (85 ± 17%) groups, but there was a decrease in the response to AII in group H/R24 since the elevation in [Ca+2]i was significantly lower than in control (61 ± 10%, p < 0.05). Glybenclamide did not modify this response. It was observed a significant increase in NO generation after 24 hours of reoxygenation, but no difference in PGE2 production was observed. Data suggest that H/R injury is characterized by increased basal [Ca+2]i and by an impairment in the response of cells to AII. Results suggest that the relative insensibility to AII may be at least in part mediated by NO but not by prostaglandins or vasodilator K ATP channels. CONCLUSION: H/R caused dysfunction in IMC characterized by increases in basal [Ca+2]i during hypoxia and reduction in the functional response to AII during reoxygenation.

Differences between in vitro and in vivo obtained schistosomules

The injection of cercariae of Schistosoma mansoni into the peritoneal cavity of naive mice induces cell adhesion to these larvae, and this adherence sharply decreases when the infecting larva changes to schistosomule. This procedure was used to detect differences between schistosomules obtained in vivo and in vitro. Reinoculation of schistosomules obtained in vivo into the peritoneal cavity of mice did not trigger cell adhesion. In contrast, adherent cells were found in 4 and 24-hour-in vitro schistosomules. Our data on schistosomules obtained in vitro indicate that more than 24 hours are needed for complete remotion of molecules involved in the phenomenon of cell adhesion.

In vitro action of some disinfectants on Paracoccidioides brasiliensis yeast forms

The fungicidal action of sodium hypochlorite (0.3, 1, 2.5, 5 and 10%); formaldehyde (2, 5, and 10%); and ethyl alcohol (70%) on yeast forms of Paracoccidioides brasiliensis Pb 18 and a newly-isolated Goiana strain was described. Contact between the fungus and the disinfectants was maintained for 1, 2, 24, 48 and 72 hours at room temperature. Viability was evaluated by the fluorescein diacetate-ethidium bromide treatment, culture in solid and liquid media (36ºC and 26ºC); yeast to mycelial germination at room temperature; and radiometric study of metabolic activity. All concentrations of disinfectants were found to be effective in inactivating Pb 18 and Goiana strains, except for the 1-hour contact with 2% formaldehyde, in which fluorescein diacetate-ethidium bromide treatment was found to reveal 40 and 27% of viable cells, respectively. The yeast to mycelial germination method was considered to reveal faster and similar results as compared to culture in solid and liquid media.

Immunoglobulin Fc receptors in clinical strains of Staphylococcus aureus do not confer resistance to Phagocytosis in an in vitro assay

Staphylococcus aureus binds Immunoglobulin G (IgG) on its external surface due to the presence of specific receptors for the Fc domain of this immunoglobulin. This mechanism represents a kind of camouflage against phagocytic cells. In order to confirm that possibility an in vitro evaluation of the phagocytic activity of leukocytes polymorpho-nuclear (PMN) against strains of Staphylococcus aureus was done, comparing 18 strains isolated from clinical samples and 16 from healthy individuals. The presence of Fc receptors was evaluated by haemagglutination (HA) with erythrocytes group A after incubation of the strains with IgG anti blood group A. Phagocytosis of S. aureus was carried out by mixing live bacteria with a suspension of human PMN and incubating at 37 °C for 1 h; survivors were counted as colony forming units by plating. The strains from clinical specimens showed higher HA than those from healthy individuals (p = 0.01); but the former were killed more efficiently than the latter (80-90% and 40%, respectively). It is may be possible that S. aureus showed different behavior in vivo, where could express other virulence factors to prevent the action of phagocytes.

Evaluation of standard reagents for radial-immunodiffusion assays. In vitro control of rabies vaccines

The RID assay is one of the in vitro methods used for in-process control in the production of rabies vaccines for veterinary use. It has been shown to be very useful for determining antigen concentration in the final bulk product. The work presented in this paper, including the production and standardization of candidate standard reagents for use in the Radial Immunodiffusion Assay (RID) was carried out at the Pan American Institute for Food Protection and Zoonoses (INPPAZ/PAHO/WHO). The study was completed with the cooperation of the Faculty of Veterinary Sciences, National University of La Plata (NULP), Argentina, where the validation of the proposed standards and the quality control of samples from 28 different batches of rabies vaccines produced with Pasteur strain rabies virus (PV) in BHK cells were performed. The activity of the vaccines was determined by in vivo (NIH) and in vitro (RID)assays. The results of the candidate reagents for the reagent standardization tests showed stability, sensitivity and reproducibility. The Relative Potency the 1.2 between the problem vaccines and the reference vaccine was estimated by variance and regression analysis. The results of our validation study show that the INPPAZ (PAHO/WHO) is capable of producing and distributing the above-mentioned standard reagents, as well as of providing support for the incorporation of the RID technique (sensitive, rapid and inexpensive) to the laboratories that manufacture rabies vaccines in Latin America and the Caribbean.

In vitro and in situ activation of the complement system by the fungus Lacazia loboi

Since there are no studies evaluating the participation of the complement system (CS) in Jorge Lobo's disease and its activity on the fungus Lacazia loboi, we carried out the present investigation. Fungal cells with a viability index of 48% were obtained from the footpads of BALB/c mice and incubated with a pool of inactivated serum from patients with the mycosis or with sterile saline for 30 min at 37 ºC. Next, the tubes were incubated for 2 h with a pool of noninactivated AB+ serum, inactivated serum, serum diluted in EGTA-MgCl2, and serum diluted in EDTA. The viability of L. loboi was evaluated and the fungal suspension was cytocentrifuged. The slides were submitted to immunofluorescence staining using human anti-C3 antibody. The results revealed that 98% of the fungi activated the CS by the alternative pathway and no significant difference in L. loboi viability was observed after CS activation. In parallel, frozen histological sections from 11 patients were analyzed regarding the presence of C3 and IgG by immunofluorescence staining. C3 and IgG deposits were observed in the fungal wall of 100% and 91% of the lesions evaluated, respectively. The results suggest that the CS and immunoglobulins may contribute to the defense mechanisms of the host against L. loboi.

The value of in vitro cell culture of granulocytes in the detection of Ehrlichia

Peripheral blood leukocytes from different animals were isolated from whole blood and maintained in Dulbeco's medium containing homologous serum without antibiotics. After 72 hrs microscopic examination of these cells showed that most animals were infected with Ehrlichia. Observation of thin blood smears from the same animals showed that only two were positive for Ehrlichia. The results of this investigation show that leukocyte culture is superior to the traditional thin blood film method in the detection of Ehrlichia and that asymptomatic carriers are easily detected. The method is inexpensive and does not require specific cell lines although it is necessary to use sterile sera.

Stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes by human monocytes

Monocytes/macrophages play a critical role in the defense mechanisms against malaria parasites, and are the main cells responsible for the elimination of malaria parasites from the blood circulation. We carried out a microscope-aided evaluation of the stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes, by human monocytes. These cells were obtained from healthy adult individuals by means of centrifugation through a cushion of Percoll density medium and were incubated with erythrocytes infected with Plasmodium falciparum that had previously been incubated with a pool of anti-plasmodial immune serum. We described the stages of phagocytosis, starting from adherence of infected erythrocytes to the phagocyte membrane and ending with their destruction within the phagolisosomes of the monocytes. We observed that the different erythrocytic forms of the parasite were ingested by monocytes, and that the process of phagocytosis may be completed in around 30 minutes. Furthermore, we showed that phagocytosis may occur continuously, such that different phases of the process were observed in the same phagocyte.

In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii

INTRODUCTION: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. METHODS: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36ºC with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. RESULTS: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075µM and an LLC MK2 toxicity of 2.003µM. Pyrimethamine was effective at an IC50 of 0.482µM and a toxicity of 11.178µM. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. CONCLUSIONS: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

In vitro detection of hepatitis C virus in platelets from uninfected individuals exposed to the virus

IntroductionDespite hepatocytes being the target cells of hepatitis C virus (HCV), viral ribonucleic acid RNA has been detected in other cells, including platelets, which have been described as carriers of the virus in the circulation of infected patients. Platelets do not express cluster differentiation 81 CD81, the main receptor for the virus in hepatocytes, although this receptor protein has been found in megakaryocytes. Still, it is not clear if HCV interacts with platelets directly or if this interaction is a consequence of its association with megakaryocytes. The aim of this study was to evaluate the interaction of HCV with platelets from non-infected individuals, after in vitro exposure to the virus.MethodsPlatelets obtained from 50 blood donors not infected by HCV were incubated in vitro at 37°C for 48h with serum containing 100,000IU∕mL of genotype 1 HCV. After incubation, RNA extracted from the platelets was assayed for the presence of HCV by reverse transcription – polymerase chain reaction RT-PCR.ResultsAfter incubation in the presence of virus, all samples of platelets showed HCV RNA.ConclusionsThe results demonstrate that, in vitro, the virus interacts with platelets despite the absence of the receptor CD81, suggesting that other molecules could be involved in this association.

Evaluation of CD4+CD25+ T lymphocyte response time kinetics in patients with chronic Chagas disease after in vitro stimulation with recombinant Trypanosoma cruzi antigens

Introduction CD4+CD25+ T lymphocytes have been implicated in the regulation of host inflammatory response against Trypanosoma cruzi, and may be involved in the clinical course of the disease. Methods Peripheral blood mononuclear cells from patients with chronic Chagas disease were cultured in the presence of T. cruzi recombinant antigens and assayed for lymphocytes at distinct time points. Results It was possible to differentiate clinical forms of chronic Chagas disease at days 3 and 5 according to presence of CD4+CD25+ T cells in cell cultures. Conclusions Longer periods of cell culture proved to be potentially valuable for prospective evaluations of CD4+CD25+ T lymphocytes in patients with chronic Chagas disease.

Paroxetine and bupropion have no in vitro effects on lynphocyte proliferation and viability

OBJECTIVE: Initial studies with tricyclic antidepressants demonstrated that they jeopardize the immune system activity. Recent studies suggested that selective serotonin reuptake inhibitors would have stimulating immunological effects. Here, we explored the in vitro immunological effects of two antidepressants used in clinical practice, paroxetine (selective serotonin reuptake inhibitor) and bupropion (norepinephrine and dopamine reuptake inhibitor). METHOD: Peripheral blood samples were obtained from 16 healthy volunteers and the peripheral blood mononuclear cells were isolated and cultured in vitro. We evaluated the effects of bupropion and paroxetine on cell viability as well as the ability to suppress phytohemagglutinin-induced lymphocyte proliferation. RESULTS: Both antidepressants produced neither significant effect on cell viability nor on T-cell proliferation. CONCLUSIONS: This could be of valuable information for the clinical practice when these drugs are administered. These results indicate a more favorable effect of such psychopharmacological drugs when compared to reported immunological effects associated with tryciclic antidepressants.